Our research program focuses on the mechanisms of retroelement action. Our approach to understanding the complex interactions between the retroelement and its host is to study retrotransposons, a family of elements that are closely related to retroviruses. A significant advantage to studying retrotransposons is they exist in hosts such as yeast that can readily be studied using sophisticated molecular genetic techniques. Using our improved in vivo transposition assay, we have collected strong evidence that Tf1 reverse transcriptase uses a novel self-priming mechanism to initiate cDNA synthesis. This is in complete contrast to the tRNA mechanisms thought to be used by all other LTR-containing elements. Analysis of other transposons indicated that the self-priming mechanism of Tf1 defines a new family of retroelements. The assembly of functional Tf1 particles has been a paradox since other retroelement particles assemble with a molar excess of capsid protein that accumulates because the levels of the Pol proteins are restricted by reading frameshifts or stop codons. Tf1 however, expresses all its protein from within a single open reading frame as a primary translation product. Because there is no obvious mechanism for overproducing Gag protein, we wondered if Tf1 particles contained equal amounts of Gag and Pol proteins or if there is a specific postranslational process that adjusts the ratios to allow for assembly. Careful quantitative measurements made this year indicate Tf1 particles contain a geater than 20-fold excess of Gag over IN. By looking at cultures in different stages of growth, we have been able to observe a degradation process that leads to this excess of Gag. To determine the factors required for transposition, we have created two large sets of mutant strains that are defective for transposition. One collection of mutants was generated by treating the host cells with a mutagen while the other set was made by creating mutations in Tf1 itself. We are now in the process of analyzing both sets and hope as a result to identify host genes that are intimately involved in transposition. Thus far, we have identified two host mutants that are defective for the degradation mechanism required to create the molar excess of Gag to Pol proteins.